Inhibitors of Multidrug Resistance Transporter P-Glycoprotein

ABSTRACT

The present disclosure provides a method of treating a subject that is resistant to one or more drugs by identifying a subject having one or more drug resistant cells; administering to the subject a pharmaceutically effective amount of an inhibitor compound, and contacting one or more drug resistant cells with the inhibitor compound to reduce the export of the inhibitor compound from the one or more drug resistant tumor cells and to block the transport of drug(s) from the one or more drug resistant cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/406,036 filed on Jan. 13, 2017 and entitled“Inhibitors of Multidrug Resistance Transporter P-Glycoprotein,” whichis a continuation application of U.S. patent application Ser. No.14/598,022 filed on Jan. 15, 2015, now U.S. Pat. No. 9,561,227, andentitled “Inhibitors of Multidrug Resistance TransporterP-Glycoprotein,” which is a non-provisional application of U.S.Provisional Patent Application No. 61/927,767, filed Jan. 15, 2014, thecontents of which are incorporated by reference in their entirety. Thisapplication is related to a continuation-in-part U.S. patent applicationSer. No. 15/911,441, filed Mar. 5, 2018, and entitled “Inhibitors ofMultidrug Resistance Transporter P-Glycoprotein.”

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under 1R15GM094771-01A1awarded by the National Institute of General Medical Sciences(NIH/NIGMS). The government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of chemotherapy,and more particularly, to the treatment of chemotherapy-resistantcancers, primary cancers and cancer stem cells, and in the field ofmodifying the blood brain barrier.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with P-glycoprotein.

A fundamental characteristic of cancerous cells that normal cells lackis the ability of the cancerous cells to sustain chronic proliferation.Uncontrolled proliferation of cells in the body often creates a seriouspathological state that requires medical intervention. The use of cancerchemotherapy began in 1948 when Sidney Farber reported that treatingpatients with a folate-dependent leukemia with an anti-folatechemotherapy could lead to temporary remissions in several children.Unfortunately, the toxic side effects of these chemotherapeutic agentsprohibited extended therapy at that time. Within 25 years, combinationsof different chemotherapeutics, tailored to specific cancers, had becomeroutine. The most successful contemporary treatments for serious cancersoften include localized treatment via surgical or radiation techniqueswhen possible, followed by systemic chemotherapies. These chemotherapiesoften involve the parenteral administration of very cytotoxic compoundsin attempts to eliminate proliferating cancer cells that remain in thebody, while trying to not affect normal cells to the same degree.

U.S. Pat. No. 7,214,664, entitled “Peptidyl prodrugs that resistP-glycoprotein mediated drug efflux” discloses dipeptide, tripeptide,and tetrapeptide ester derivatives of bioactive agents that aresubstrates effluxed by the P-glycoprotein transporter. The derivativesare said to be useful in treating the same condition as the bioactiveagent and a method for preparing a bioactive agent for targeted deliveryby nutrient or peptide transporters comprising linking the agent to oneor more groups of the formula —X—Y_(n)—Z_(n′)—Z′_(n″)—R; wherein each X,Y, Z, and Z′ is independently Met, Val, Thr, Tyr, Trp, Ser, Ala or Gly;R is independently H or an amino-protecting group; n=1, and each, n′, orn″ is independently 0 or 1.

U.S. Pat. Nos. 7,144,704; 6,630,327; 6,365,357; and 5,994,088, relatedto methods and reagents for preparing and using immunological agentsspecific for P-glycoprotein are directed to immunological reagents andmethods specific for a mammalian, transmembrane P-glycoprotein, which issaid to be a non-specific efflux pump activity, and isclinically-important in multidrug resistance in cancer patientsundergoing chemotherapy. They disclose methods for developing and usingimmunological reagents specific for certain mutant forms ofP-glycoprotein and for wild-type P-glycoprotein in a conformationassociated with substrate binding, or in the presence of ATP depletingagents, provide improved methods for identifying and characterizinganticancer compounds.

U.S. Pat. No. 5,763,443, entitled “MDR resistance treatment and novelpharmaceutically active riminophenazines” and discloses the use ofriminophenazines in the treatment of a patient who has built up, orcould build up, resistance to a therapeutically active substance, suchas a patient requiring treatment for cancer, and includes novelriminophenazines, their preparation, and compositions containing them.

United States Patent Application Publication No. 2010/0068786, entitled“Methods and compositions for reversing P-glycoprotein medicated drugresistance,” discloses a method for inhibiting therapeutic drugresistance within a cell over-expressing a membrane protein is provided,wherein the method comprises synthesizing a dimeric prodrug inhibitor ofa monomeric therapeutic agent; administering the dimeric prodruginhibitor to the membrane protein together with the monomerictherapeutic agent; and occupying at least one substrate binding site ofthe membrane protein with the synthesized dimeric prodrug to allow themonomeric therapeutic agent to accumulate within the cell. The dimericprodrug inhibitor contains a crosslinking agent that is adapted tobreakdown under reducing conditions within the cytosol of the cell tocause the dimeric prodrug to revert back to a form equivalent to themonomeric therapeutic agent.

Multidrug resistance is a significant problem in the pharmaceuticalindustry, and may be achieved by the activation of cellular membranetransporters. Drugs and certain proteins are transported across themembranes by energy-activated pumps where the outer membrane componentof these pumps is a channel that opens from a sealed resting stateduring the transport process. For example, exporter proteins confer drugresistance by pumping the drug out of the cell before the drug canfunction or exert its intended effect (e.g., kill a cancer cell).Classical inhibitors of the exporter proteins are bulky hydrophobicmolecules that overload the capacity of the hydrolysis of two ATPmolecules to expel the drug by disruption of the hydrophobicassociations, but these inhibitors lack specificity and are associatedwith significant side effects, disrupting important functions in tissuesthroughout the body.

U.S. Pat. No. 8,626,452, entitled “Compositions and methods foroptimizing drug hydrophobicity and drug delivery to cells,” disclosesmethods to determine drug hydrophobicity and to quantify changes in drughydrophobicity that optimize drug function by means of differentialscanning calorimetry of an endothermic phase transition of a baseprotein-based polymer, specifically of an elastic-contractile modelprotein, to which is attached to the drug to be evaluated for itshydrophobicity in terms of the change in Gibbs free energy forhydrophobic association have been developed. Also described is thepreparation of nanoparticles comprised of protein-based polymers,specifically of elastic-contractile model proteins, designed for thebinding and desired release rate of a specific drug or class of drugs.Further described is a means of targeting the drug-laden nanoparticle toa cell by means of decorating the nanoparticle surface with a molecularentity that selectively binds to the diseased cell or disease causingorganism, e.g., by decorating the drug-laden nanoparticle surface withsynthetic antigen-binding fragment to an up-regulated receptorcharacteristic of the diseased cell.

SUMMARY OF THE INVENTION

The present disclosure provides a method of treating a subject having acancer that is resistant to one or more chemotherapeutic drugs byidentifying a subject having one or more drug resistant cancer cells;administering to the subject a pharmaceutically effective amount of aninhibitor compound having one of the following structural formulas:

andcontacting one or more drug resistant cancer cells with the inhibitorcompound to reduce the drug resistance of the cancer cells. In oneaspect, the inhibitor compound can reduce the export of the inhibitorcompound from the one or more drug resistant tumor cells and/or blockthe transport of chemotherapeutic drug(s) from the one or more drugresistant cancer cells. In one aspect, the inhibitor compound interactswith an exporter protein. In another aspect, inhibitor compound is aP-glycoprotein inhibitor. In another aspect, the inhibitor compoundinteracts with drug-toxin pumping structures of a P-glycoprotein. Inanother aspect, the inhibitor compound interacts with ATP bindingdomain(s) of a P-glycoprotein and the inhibitor compound does not bindto drug binding site(s) on the P-glycoprotein. In another aspect, theinhibitor compound is minimally transported by a P-glycoprotein. Inanother aspect, the one or more drug resistant cancer cells are one ormore multidrug resistant tumor cells. In another aspect, the one or moredrug resistant cancer cells are lymphoma, leukemia, cancer stem cells,nonsmall-cell lung cancer, liver cancer, encephaloma, leukocythemia,carcinoma of prostate, intestine cancer, myeloma tumor, lymphoma, breastcarcinoma, ovarian cancer, gastric cancer, small cell lung cancer,esophageal carcinoma, esophageal carcinoma, and sarcoma. In anotheraspect, further comprises the step of administering one or morechemotherapeutic agents to the subject.

The present disclosure provides a method of sensitization andre-sensitization of a cancer cell to a chemotherapeutic by identifying asubject having one or more cancer cells in need of sensitization andre-sensitization to one or more chemotherapeutics; administering to thesubject a pharmaceutically effective amount of an inhibitor compoundhaving one of the following structural formulas:

andcontacting one or more cancer cells with the inhibitor compound toreduce the export of the inhibitor compound from the one or more cancercells and to block the transport of the one or more chemotherapeuticdrug(s) from the one or more cancer cells to sensitization orre-sensitization of the one or more cancer cells to the one or morechemotherapeutic drug(s). In one aspect, the inhibitor compoundinteracts with an exporter protein. In another aspect, inhibitorcompound is a P-glycoprotein inhibitor. In another aspect, the inhibitorcompound interacts with drug-toxin pumping structures of aP-glycoprotein. In another aspect, the inhibitor compound interacts withATP binding domain(s) of a P-glycoprotein and the inhibitor compounddoes not bind to drug binding site(s) on the P-glycoprotein. In anotheraspect, the inhibitor compound is minimally transported by aP-glycoprotein. In another aspect, the one or more drug resistant cancercells are one or more multidrug resistant tumor cells. In anotheraspect, the one or more drug resistant cancer cells are lymphoma,leukemia, cancer stem cells, nonsmall-cell lung cancer, liver cancer,encephaloma, leukocythemia, carcinoma of prostate, intestine cancer,myeloma tumor, lymphoma, breast carcinoma, ovarian cancer, gastriccancer, small cell lung cancer, esophageal carcinoma, esophagealcarcinoma, and sarcoma. In another aspect, further comprises the step ofadministering one or more chemotherapeutic agents to the subject.

The present disclosure also provides a method of increasing an efficacyof one or more chemotherapeutics and/or decreasing toxicity of one ormore chemotherapeutic treatments by identifying a subject having one ormore cancer cells; administering to the subject one or morechemotherapeutics; administering to the subject a pharmaceuticallyeffective amount of an inhibitor compound having one of the followingformulas:

andcontacting one or more cancer cells with the inhibitor compound toreduce the export of the inhibitor compound from the one or more cancercells and to block the transport of the one or more chemotherapeuticsfrom the one or more cancer cells to increasing the efficacy of the oneor more chemotherapeutics and/or decreasing toxicity of the one or morechemotherapeutic treatments.

The present disclosure provides a method of increasing the penetrationof an agent through the blood brain barrier or the blood testis barrierby identifying a subject in need of increasing the penetration of anagent through the blood brain barrier or the blood testis barrier;administering to the subject a pharmaceutically effective amount of aninhibitor compound having one of the following formulas:

andwherein the inhibitor compound reduces the export of the inhibitorcompound from the one or more cells and increases the penetration of theagent through the blood brain barrier or the blood testis barrier.

The present disclosure provides a method of decreasing theP-glycoprotein activity of a cancer patient who has built up resistanceto a therapeutically active agent used in the treatment of cancer toreduce the resistance to further treatment with the substance byadministering to a cancer patient before further treatment, duringtreatment or after treatment with the therapeutically active agent, anamount of an inhibitor compound having one of the following structuralformulas:

andto reduce the export of the inhibitor compound, and to block thetransport of the therapeutically active agent from one or more cancercells of the cancer patient.

The present disclosure provides a method of reducing the likelihood ofdeveloping a resistance to a therapeutically active agent byadministering to a patient before further treatment, during treatment orafter treatment with the therapeutically active agent, an amount of aninhibitor compound having one of the following structural formulas:

andto reduce the export of the inhibitor compound and to block thetransport of the therapeutically active agent from one or more cells ofthe patient.

The present disclosure provides a method for modulating the activity ofa cell membrane transporter in a biologic tissue by contacting a tissuehaving a cell membrane transporter with a pharmaceutically effectiveamount of an inhibitor compound having one of the following structuralformulas:

The present disclosure provides a method for treating a disease orcondition in a mammal or avian resulting from an activity of a cellmembrane transporter by administering to said mammal or avian atherapeutically effective amount of a pharmaceutical composition havingthe structural formulas:

or pharmaceutically acceptable salts thereof and a pharmaceuticallyacceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying FIGS. and in which:

FIGS. 1A-1D are plots showing the inhibition of ATP hydrolysis by insilico identified compounds. The FIG. shows the concentration dependenceof inhibition of the four identified inhibitor compounds (abbreviated19, 29, 34 and 45). Each plot represents the composite results of 3 to 5individual studies performed on three different P-glycoproteinpreparations.

FIGS. 2A-2D are plots showing the effects of P-glycoprotein ATPhydrolysis inhibitors on nucleotide binding. Between 30 and 50 microMP-glycoprotein were incubated with 50 microM inhibitor compound.Increasing amounts of SL-ATP were added. The ESR signal of the freeSL-ATP in the presence of P-glycoprotein and inhibitor was compared tothat in the absence of P-glycoprotein. The graphs represent at least 3individual studies each using 3 to 5 different P-glycoproteinpreparations.

FIGS. 3A-3B are plots that show an example of the re-sensitization of amultidrug resistant prostate cancer cell line to two differentchemotherapeutics using the prostate cancer derived cell line, DU145 andthe multidrug-resistant variant, DU145-TxR.

FIGS. 4A and 4B are graphs that show the in silico identified P-gpinhibitors potentiate the cytotoxic effects of paclitaxel in the MDRhuman prostate cancer cell line DU145TxR. Cells were incubated with 50nM-25 μM inhibitor 19 (diamonds), 29 (squares), 34 (triangles), 45(inverted-triangles), or verapamil (octagons) with 500 nM paclitaxel(top) or without paclitaxel (bottom) for 48 hours and survival wasdetermined by MTT assay as previously described. Values represent themean±SEM of at least two separate experiments performed in triplicate.

FIGS. 5A to 5C are graphs that show the intrinsic toxicities ofexperimental compounds: The effects of compounds 19 (diamonds), 29(squares), 34 (triangles), 45 (inverted triangles) and verapamil(octagons) on the survival of (5A) noncancerous HFL1, (5B) prostatecancer cell line, DU145, (5C) MDR prostate cancer cell line, DU145TxR.Percent survival determined by MTT assay calculated relative to cellstreated with equal volume DMSO vehicle and represent the mean±SEM from2-4 separate experiments, each experiment performed in triplicate wells.

FIGS. 6A to 6C are graphs that show dose dependent sensitization of MDRprostate cancer cell line DU145TxR. DU145TxR cells (closed symbols) wereincubated with various concentrations of paclitaxel and 5 μM (6A), 10 μM(6B), or 25 μM (6C) in silico identified P-gp inhibitors 19 (diamonds),29 (squares), 34 (triangles), 45 (inverted triangles) and verapamil(octagons). For reference, the DU145TxR (closed circle) and DU145 (opencircle) incubated with paclitaxel only are also included. Values aremean±SEM from at least 2 separate experiments performed in triplicatewells. The data were normalized to the intrinsic toxicities asdetermined in experiments from FIG. 5 of each of the compounds for eachof the corresponding concentrations.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

As used herein, the term “alkyl” denotes optionally substituted straightchain and branched hydrocarbons having about 1 to about 50 carbons withat least one hydrogen removed to form a radical group. Alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,1-methylpropyl, pentyl, isopentyl, sec-pentyl, hexyl, heptyl, octyl, andso on. Alkyl includes cycloalkyl, such as cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl.

As used herein, the term “alkenyl” includes optionally substitutedstraight chain and branched hydrocarbon radicals having about 1 to about50 carbons as above with at least one carbon-carbon double bond (sp2).Alkenyls include ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (orallyl), isopropenyl (or 1-methylvinyl), but-1-enyl, but-2-enyl,butadienyls, pentenyls, hexa-2,4-dienyl, and so on. Hydrocarbon radicalshaving a mixture of double bonds and triple bonds, such as2-penten-4-ynyl, are grouped as alkynyls herein. Alkenyl includescycloalkenyl. Cis and trans or (E) and (Z) forms are included within theinvention.

As used herein, “Alkoxy” refers to the group “alkyl-O—” which includes,by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, andthe like. As used herein, the term “alkoxy” includes an optionallysubstituted straight chain or branched alkyl group having about 1 toabout 50 carbons with a terminal oxygen linking the alkyl group to therest of the molecule. Alkoxy includes methoxy, ethoxy, propoxy,isopropoxy, butoxy, t-butoxy, pentoxy and so on. “Aminoalkyl”,“thioalkyl”, and “sulfonylalkyl” are analogous to alkoxy, replacing theterminal oxygen atom of alkoxy with, respectively, NH (or NR), S, andSO2. Heteroalkyl includes alkoxy, aminoalkyl, thioalkyl, and so on. Theterm “alkoxy” denotes —OR—, wherein R is alkyl.

As used herein, “Substituted alkoxy” refers to the group “substitutedalkyl-O—”.

As used herein, the term “alkylcarbonyl” denote an alkyl group of theformula —C(O)Rc wherein Rc is alkoxy, substituted with a C(O) group, forexample, CH3C(O)—, CH₃CH₂C(O)—, etc.

As used herein, “Alkanoate” refers to “alkyl-C(═O)—O—” which includes,by way of example, ethanoate and pentanoate. “Alkyl-Alkanoate” refers to“-alkyl-O—C(═O)alkyl” such as in —CH(CH₂CH₃)—O—C(═O)—CH₃.

As used herein, “Alkylcarbonylalkoxy” refers to alkyl-C(═O)—O-alkyl. Inone variation, the alkylcarbonylalkoxy refers to a moiety C₁-C₄alkyl-C(═O)—O—C₁-C₆ alkyl. An exemplary alkylcarbonylalkoxy is—CH₂CH₂C(═O)OCH₃.

As used herein, the term “alkylcarboxyl” denote an alkyl group asdefined above substituted with a C(O)O group, for example, CH₃C(O)O—,CH₃CH₂C(O)O—, etc.

As used herein, “Carbonylalkyl” refers to —C(═O)-alkyl, which includes,by way of example, —C(═O) CH₂CH₃.

As used herein, the term “carboxy” refers to the group —C(O)OH.

As used herein, the term “carboxyl” denotes —C(O)O—, and the term“carbonyl” denotes —C(O)—.

As used herein, “C₃-C₈ monocyclic cycloalkyl” as used herein is a 3-,4-, 5-, 6-, 7- or 8-membered saturated non-aromatic monocycliccycloalkyl ring. Representative C₃-C₈ monocyclic cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl. In one embodiment, the C₃-C₈monocyclic cycloalkyl group is substituted with one or more of thefollowing groups: -halogen, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′,—OC(O)R′, —N(R)₂, —NHC(O)R′ or —C(O)NHR′ groups wherein each R′ isindependently —H or unsubstituted —C₁-C₆ alkyl. Unless indicated, theC₃-C₈ monocyclic cycloalkyl is unsubstituted.

As used herein, “C₃-C₈ monocyclic cycloalkenyl” as used herein is a 3-,4-, 5-, 6-, 7- or 8-membered non-aromatic monocyclic carbocyclic ringhaving at least one endocyclic double bond, but which is not aromatic.It is to be understood that when any two groups, together with thecarbon atom to which they are attached form a C₃-C₈ monocycliccycloalkenyl group, the carbon atom to which the two groups are attachedremains tetravalent. Representative C₃-C₈ monocyclic cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,1,3-cyclobutadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl,1,3-cyclohexadienyl, cycloheptenyl, 1,3-cycloheptadienyl,1,4-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctenyl,1,3-cyclooctadienyl, 1,4-cyclooctadienyl, or 1,3,5-cyclooctatrienyl. Inone embodiment, the C₃-C₈ monocyclic cycloalkenyl group is substitutedwith one or more of the following groups: -halo, —O—(C₁-C₆ alkyl), —OH,—CN, —COOR′, —OC(O)R′, —N(R)₂, —NHC(O)R′ or —C(O)NHR′ groups whereineach R′ is independently —H or unsubstituted C₁-C₆ alkyl. Unlessindicated, the C₃-C₈ monocyclic cycloalkenyl is unsubstituted.

The term “3- to 7-membered monocyclic heterocycle” refers to: a 3-, 4-,5-, 6-, or 7-membered aromatic or non-aromatic monocyclic cycloalkyl inwhich 1-4 of the ring carbon atoms have been independently replaced withan NH, an O, or an S moiety. The non-aromatic 3- to 7-memberedmonocyclic heterocycles can be attached via a ring nitrogen, sulfur, orcarbon atom. The aromatic 3- to 7-membered monocyclic heterocycles areattached via a ring carbon atom. Representative examples of a 3- to7-membered monocyclic heterocycle group include, but are not limited tofuranyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl,oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,piperazinyl, piperidinyl, pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole,pyridothiazole, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,quinuclidinyl, tetrahydrofuranyl, thiadiazinyl, thiadiazolyl, thienyl,thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiomorpholinyl,thiophenyl, triazinyl, triazolyl, In one embodiment, the 3- to7-membered monocyclic heterocycle group is substituted with one or moreof the following groups: -halo, —O—(C₁-C₆ alkyl), —OH, —CN, —COOR′,—OC(O)R′, —N(R′)₂, —NHC(O)R′ or —C(O)NHR′ groups wherein each R′ isindependently —H or unsubstituted C₁-C₆ alkyl. Unless indicated, the 3-to 7-membered monocyclic heterocycle is unsubstituted.

The phrases “therapeutically effective amount” and “effective dosage”denotes an amount sufficient to produce a therapeutically (e.g.,clinically) desirable result; the exact nature of the result will varydepending on the nature of the disorder being treated. For example,where the disorder to be treated is cancer, the result can be thereduction of cancerous cells including cancerous tumors or theamelioration of symptoms related to the cancer cells. The compositionsdescribed herein can be administered from one or more times per day toone or more times per week. The skilled artisan will appreciate thatcertain factors can influence the dosage and timing required toeffectively treat a subject, including but not limited to the severityof the disease or disorder, previous treatments, the general healthand/or age of the subject, and other diseases present. Moreover,treatment of a subject with a therapeutically effective amount of thecompositions of the invention can include a single treatment or a seriesof treatments.

As used herein, the term “treatment” denotes the application oradministration of a therapeutic agent described herein, or identified bya method described herein, to a patient, or application oradministration of the therapeutic agent to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease, or the predisposition toward disease.

The term “optional” or “optionally” denotes that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns that are stericallyimpractical and/or synthetically non-feasible.

As used herein, the terms “patient,” “subject” and “individual” are usedinterchangeably herein, and denote a mammalian subject to be treated,with human patients being preferred. In some cases, the methods of theinvention find use in experimental animals, in veterinary applications,and in the development of animal models for disease.

As used herein, the term “chemotherapeutic agent” includes agents suchas drugs which can advantageously be administered to the tissue, such asanti-tumor drugs such as paclitaxel, doxorubicin, and other drugs whichhave been known to affect tumors. It also includes agents which modulateother states which are related to tissues which can be permeabilizedusing the methods and compositions of the invention. Thechemotherapeutic agent can be, for example, a steroid, an antibiotic, oranother pharmaceutical composition. Examples of chemotherapeutic agentsinclude agents such as paclitaxel, doxorubicin, vincristine,vinblastine, vindesine, vinorelbin, taxotere (DOCETAXEL), topotecan,camptothecin, irinotecan hydrochloride (CAMPTOSAR), doxorubicin,etoposide, mitoxantrone, daunorubicin, idarubicin, teniposide,amsacrine, epirubicin, merbarone, piroxantrone hydrochloride,5-fluorouracil, methotrexate, 6-mercaptopurine, 6-thioguanine,fludarabine phosphate, cytarabine (ARA-C), trimetrexate, gemcitabine,acivicin, alanosine, pyrazofurin, N-Phosphoracetyl-L-Asparate (PALA),pentostatin, 5-azacitidine, 5-Aza-2′-deoxycytidine, adenosinearabinoside (ARA-A), cladribine, ftorafur, UFT (combination of uraciland ftorafur), 5-fluoro-2′-deoxyuridine, 5-fluorouridine,5′-deoxy-5-fluorouridine, hydroxyurea, dihydrolenchlorambucil,tiazofurin, cisplatin, carboplatin, oxaliplatin, mitomycin C, BCNU(Carmustine), melphalan, thiotepa, busulfan, chlorambucil, plicamycin,dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen mustard,uracil mustard, pipobroman, 4-ipomeanol, dihydrolenperone, spiromustine,geldanamycin, cytochalasins, depsipeptide, Lupron, ketoconazole,tamoxifen, goserelin (Zoledax), flutamide,4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide, Herceptin, anti-CD20 (Rituxan), interferon alpha,interferon beta, interferon gamma, interleukin 2, interleukin 4,interleukin 12, tumor necrosis factors, and radiation.

As used herein, the term “delivering” denotes making the compositionavailable to the interior of the tissue (e.g., cancer cells, tumor,bacterial cell, etc.) to be treated such that the composition is capableof having a therapeutic effect on the interior of the tissue andincludes, for example, contacting the tissue with the agent. The term“delivering” is intended to include administering the composition to thepatient as a separate dose, as well as administering the composition tothe patient together with (i.e., at the same time as or in the same doseas) other agents.

As used herein, the terms “proliferative disorder”, “hyperproliferativedisorder,” and “cell proliferation disorder” are used interchangeably tomean a disease or medical condition involving pathological growth ofcells. Such disorders include cancer.

As used herein, the terms “Cytotoxic agents” refer to compounds whichcause cell death primarily by interfering directly with the cell'sfunctioning or inhibit or interfere with cell mitosis, includingalkylating agents, tumor necrosis factors, intercalators, microtubulininhibitors, and topoisomerase inhibitors. Examples of cytotoxic agentsinclude, but are not limited thereto, cyclophosphamide ifosfamide,hexamethylmelamine, tirapazimine, sertenef, cachectin, ifosfamide,tasonermin, lonidamine, carboplatin, mitomycin, altretamine,prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin,oxaliplatin, temozolomide, doxorubicin heptaplatin, estramustine,improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride,pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven,dexifosfamide, cis-aminedichloro(2-methyl-pyridine) platinum,benzylguanine, glufosfamide, GPX100, (trans, trans,trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(c-hloro)-platinum(II)]tetrachloride, diarizidinylspermine, arsenic trioxide,1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin,idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin,pinafide, valrubicin, amrubicin, antineoplaston,3′-deamino-3′-morpholino-13-deoxo-10-hydroxycaminomycin, annamycin,galarubicin, elinafide, MEN10755, and4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin. “mTORinhibitors” are a subset of the cytotoxic agents and refer particularlyto inhibitors of the mTOR-Raptor complex. Included in the definition ofmTOR inhibitors are anti-cancer agents such as rapamycin and itsderivatives, sirolimus, temsirolimus, everolimus, zotarolimus anddeforolimus. Examples of microtubulin inhibitors include paclitaxel(TAXOL®), vindesine sulfate,3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxel, rhizoxin,dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881,BMS184476, vinflunine, cryptophycin,2,3,4,5,6-pentafluoro-N-(-3-fluoro-4-methoxyphenyl)benzene sulfonamide,anhydrovinblastine,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butyla-mide,TDX258, and BMS 188797. Some examples of topoisomerase inhibitors aretopotecan, hycaptamine, irinotecan, rubitecan,6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin,9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H)propanamine,1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12Hbenzo[de]pyrano[3′,4′:b,7]indolizino[1,2b]quinoline-10,13(9H,15H) dione,lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350,BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane,2′-dimethylamino-2′-deoxy-etoposide, GL331,N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazo-le-1-carboxamide,asulacrine, (5a, 5aB,8aa,9b)-9-[2-[N-[2-(dimethylamino)-ethyl]-N-methylamino]ethyl]-5-[4-Hydro-xy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,-9-hexohydrofuro(3′,′:6,7)naphtho(2,3-d)-1,3-dioxol-6-one,2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridiniu-m,6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione,5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-py-razolo[4,5,1-de]acridin-6-one,N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethy-1]formamide,N-(2-(dimethylamino)ethyl)acrid-ine-4-carboxamide,[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2-,1-c]quinolin-7-o-ne,and dimesna.

As used herein, the term “tumor” denotes abnormally growing tissue ofany tissue type and includes both benign and malignant tumors, such ascancerous tumors. Examples of cancerous tumors include sarcomas,carcinomas, adenocarcinomas, lymphomas, and leukemias.

The cancerous tumor may comprise metastatic lesion. It also includes anyother tumors which can be advantageously treated using the methods andcompositions of the invention. The cancerous tumor may be, for example,a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer,colon carcinoma, rectal cancer, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, uterine cancer, cancer of the head andneck, skin cancer, brain cancer, squamous cell carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, lung carcinoma, small cell lung carcinoma; non-small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma;hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi'ssarcoma.

Cellular membranes form a selective permeability barrier to ions andmolecules thereby maintaining intracellular concentrations that arecompatible with and optimized for physiological processes. Thispermeability barrier is comprised of a lipid bilayer, which restrictsthe passage of polar, charged and hydrophilic molecules and ions, and ofcell membrane transporters. Cell membrane transporters includeproteinaceous structures that open in a controlled manner to allowselected ions or small molecules to flow passively into or out of thecell (e.g., ion channels), other proteins that allow the facilitateddiffusion of larger polar molecules down their concentration gradients(e.g. facilitated transport of glucose), and proteins that actively pumpions and molecules into or out of the cell against a concentrationgradient (e.g., H⁺/K⁺ ATPases, MDR efflux pumps, neurotransmittertransporters).

Ion channels serve a variety of important cellular functions, includingexcitability, neuronal signaling, excitation-secretion coupling, volumeregulation and so on and are formed by the association of integralmembrane proteins into structures having a central hydrophilic pore.Channel pores allow ions to equilibrate across membranes in response totheir electrochemical gradients and at rates that are diffusion-limitedand are characterized by their selectivity and gating properties.Selectivity refers to the rate at which different ion species passthrough an open channel under standard conditions. Gating is the processthat regulates the opening and closing of an ion channel. Thus,voltage-regulated ion channels respond to changes in membrane potential;ligand-regulated channels respond to the binding of particularneurotransmitters or intracellular messengers; and mechanosensitivechannels respond to mechanical deformation. Ion channels exist inresting (closed), open or inactivated (i.e., desensitized) states.Voltage-gated ion channels in the open state typically transition to aninactivated state, and must reacquire the ability to respond to anexternal stimulus during a recovery period. This may also be true ofligand-gated channels, particularly after prolonged exposure to anagonist. Certain channels are gated by more than one type of stimulus(e.g., an inward rectifying voltage-regulated potassium channel incardiac muscle is activated by acetylcholine).

The ABC transporters comprise a superfamily that shares a highlyconserved ATP-binding cassette. These transporters typically use ATPhydrolysis as a source of energy to pump diverse classes of molecules(e.g., sugars, peptides, inorganic ions, amino acids, oligopeptides,polysaccharides, proteins) across membranes against a concentrationgradient. Some transporters are highly selective for a particularsubstrate and pump unidirectionally, and some members of the ABCtransporter family have ion channel activity. For example, the cysticfibrosis transmembrane regulator (CFTR), a cAMP- and protein kinaseA-regulated Cl⁻ channel, uses ATP hydrolysis as a gating mechanism.P-glycoprotein (MDR) appears to be bifunctional, possessing drugtransport as well as chloride channel activities; the latter iscell-volume regulated and requires the binding, but not the hydrolysis,of ATP. In both prokaryotes and eukaryotes, ABC transporters function innutrient uptake, protein export and drug resistance (e.g., erythromycinresistance in Staphylococcus, daunomycin resistance in Streptomyces,chloroquine resistance in Plasmodium, and multidrug resistance incancers). Ion pumps are also involved in the active transport of ionsacross membranes. Ion pumps can be members of the ion-transportingP-type ATPase family which couple ion transport to a cycle ofphosphorylation and dephosphorylation of an ATPase enzyme. In mammaliancells, this class includes the Ca²⁺ ATPases, the Na⁺/K⁺ ATPases, and theH⁺/K⁺ ATPases; however, V-ATPase is not a member of the ion-transportingP-type ATPase family. The H⁺/K⁺ ATPases are involved in acid secretionin the stomach, and are clinically important targets in peptic ulcerdisease, gastroesophageal reflux disease (GERD) and gastrichyperacidity. The Ca⁺² and Na⁺/K⁺ ATPases are therapeutic targets in thetreatment of heart failure.

Cancer and cancer chemotherapies. For blood cell cancers like lymphomasand leukemias, chemotherapies remain the primary methods of treatment.At the current time there are about 100 different chemotherapeutic drugsthat have been approved for use against cancers in humans in the UnitedStates. Different drugs or different combinations of drugs are beingused against different cancers. Chemotherapies have proven extremelyvaluable in many cases. For example, childhood cases of acutelymphocytic leukemia, a cancer of the blood that presents withuncontrolled multiplication of a type of white blood cells, once averitable death sentence, are now often curable.

Unfortunately, such positive outcomes are not always achieved. In fact,cancer remains the third leading cause of death, despite many advancesin recent years. One problem is that many times, after what appears tobe a successful chemotherapy, the cancer recurs and is again detected inthe patient. Such chemotherapy failures may have several causes. Onemajor cause is that a family of proteins that remove thechemotherapeutic drugs from subpopulations of the cancerous cells isoverly produced (i.e., “over-expressed”) in these cells. These proteinsare members of a class of membrane bound transporter proteins, calledABC transporters that act like cellular vacuum cleaners. They are aptlycalled multidrug resistance proteins or MDR pumps. The over-expressionof one specific member of these MDR pumps, a protein calledP-glycoprotein, seems to be especially important for cancers that becomeresistant to chemotherapeutics. Other proteins that are closely relatedto the P-glycoprotein, are also thought to be involved in otherchemotherapy failures.

P-Glycoprotein normally functions as a biochemical transporter that isable to bind a great variety of toxic chemicals inside of the livingcell and, using a chemical power source called ATP, pushes the toxinthrough the cell membrane. This action effectively removes the toxinfrom the cells. The pump is therefore an essential component inimportant tissue boundaries like the blood-brain and blood-testisbarriers. It is also helpful for the detoxification of important tissuesand organs like breast, ovaries and kidneys. While this normally veryimportant function of P-glycoprotein keeps cellular toxins at a lowconcentration in our cells and tissues, it is thought to be one of theroot causes for cancer chemotherapy failures. For example, if a subsetof cancer cells produces enough of this protein to cause the effectiveconcentration of cancer chemotherapeutic(s) inside the cancerous cellsto fall below the threshold(s) for clinical efficacy, then members ofthis subset of cancerous cells will survive the therapy. Oncemultiplying, this subset of cancerous cells will form a recurrent orrelapsed cancer that consists of or contains cells that are resistant tochemotherapeutic drugs.

Because toxin pumps like the P-glycoprotein have evolved to export awide variety of cellular toxins from the body's cells, they do notexhibit specificity towards a particular chemical compound or toxin andare capable of removing a wide variety of different toxins, drugs andchemotherapeutic compounds from the inside of the cell. Because of thislatter property, when P-glycoprotein or one of its close relatives arethe cause of chemotherapy failure, the recurrent cancer becomesresistant to many of the chemotherapeutics available, not just theone(s) used during original therapy. These recurrent cancers are now“multidrug resistant.”

Typically, a small percentage of the original cancer cells developmutations that instruct these few cells to produce more than a normalcomplement of P-glycoprotein. During chemotherapy, all or nearly all ofthe cancer cells that express low or moderate amounts of P-glycoproteinare killed, but the cells that produce high amounts of P-glycoproteinsurvive. These survivors then multiply over time and the cancer growsback. What is different, however, is that all of the cells of therecurring cancer are now over-expressing P-glycoprotein transportactivity and are therefore resistant to nearly all chemotherapies.

P-glycoprotein was linked as a cause of chemotherapy resistances incancer nearly 30 years ago. Scientists have been searching in vain forsmall molecule inhibitors of this transporter that would serve todecrease the active export of chemotherapeutics from resistant cancercells. The inhibition of P-glycoprotein of the present invention servesto re-sensitize the resistant cells to new rounds of chemotherapy,rendering the ineffective chemotherapies once more effective.

Most often in the past, inhibitors of P-glycoprotein have been foundthat interacted with the drug transporting parts of the protein. Bycompeting with the chemotherapeutic for binding to and being transportedby the pump, these inhibitors slow down the export of thechemotherapeutic drug. The problem with these types of P-glycoproteininhibitors is that high concentrations of inhibitors are then requiredto get enough inhibitor into the cell to effectively slow or stopP-glycoprotein if the inhibitors are transport substrates themselves.With the required high concentrations of inhibitors come additional orworsened side-effects caused by the P-glycoprotein inhibitorsthemselves. These side effects have led to the failure of the previouslyidentified inhibitors in clinical trials and have led to problems withtheir translation into therapeutic regimens. Thus, the long felt need isunresolved in spite of numerous attempts.

Stem cells are an important subset of the cells in all multicellularorganisms and have been found in a variety of mammalian tissues. Thesecells are of paramount importance for regeneration and repair of damagedtissues since they can either self-renew or differentiate to replacedamaged cells. Importantly, stem cells display a property of durableself-renewal or the ability to divide and reproduce many times withoutdifferentiation into a mature cell type.

Cancer stem cells (CSCs) have been defined as stem cell like cellswithin a population of cancerous cells that can renew and propagate thecancer; much like normal stem cells can propagate and renew cells ofnormal tissues. A key difference of course is that the cancer stem cellshave the acquired mutations and chromosomal alterations that lead to theuncontrolled proliferation of the cancer. Some researchers have notedthat the durability of the reservoir of cells that compose a normal orcancer stem cell population needs a constant and high level ofexpression of multidrug transporter proteins to maintain a protectedstate of the cell, i.e. a state that makes it unlikely that the stemcells will be killed by incidental chemical exposure. In recent years,this constant over-expression of P-glycoprotein in cancer stem cells hasindeed been found to be the case.

One of the great problems that cancer stem cells present is that even ifthe vast majority of a cancerous population of cells is sensitive tochemotherapeutics and is killed by the chemotherapy, the fact thatmultidrug transporters like P-glycoprotein are highly expressed in thecancer stem cell population allows these cells to survive thechemotherapy. They will ultimately renew and repopulate the cancer. Thisscenario presents the physician and patient with a seemingly initialsuccess of the therapy, followed ultimately by a recurrence of thecancer, a cancer that now consists mostly of cells that are resistant tochemotherapy.

Effective inhibition of P-glycoprotein in the context of initial cancerchemotherapy is therefore likely to also make the cancer stem cellpopulation more sensitive to the cell toxicity of the chemotherapeutic.Co-administering of P-glycoprotein inhibitors with the chemotherapeuticmay therefore be a method of treating not only drug resistant cancersbut also leads to the diminution of the cancer stem cell populationwithin the patient. This result strongly increases the therapeutic indexof the chemotherapy by preventing recurrence through the cancer stemcell population route.

The blood brain barrier (BBB) which serves to separate the blood fromthe extracellular fluid of the brain and spinal cord, i.e. centralnervous system (CNS), is created by a special capillary cellconstruction found only in these organs. The cells of the circulatorysystem in other tissues are normally not tightly associated, whichallows a much freer diffusion of substances out of the blood capillaryand into the tissues of the neighboring organ. In the brain and spinalcord, however, the endothelial cells that make up the capillary vesselsare connected by tight junctions that prohibit such free diffusionbetween cells. For compounds that are larger than small hydrophobicmolecules like oxygen and carbon dioxide, to enter the brain, they mustdiffuse into, across, and out of the endothelial barrier cells that makeup the capillary. These cells, however, express a large amount ofP-glycoprotein in the luminal membranes. This severely inhibits movementof drugs into the brain, since P-glycoprotein transports them back intothe lumen of the capillary as soon as they appear in the cytoplasm ofthe capillary cells. P-glycoprotein therefore represents an essentialpart of the BBB system, and is responsible for the poor brainpenetration of many important pharmacotherapeutics. These properties ofthe BBB, most especially the high expression of P-glycoprotein in thecells making it up, severely decrease the utility of therapeutics whenthe target organ is part of the CNS.

Numerous examples of the problems presented by P-glycoprotein in gettingeffective therapies into the brain have been reported showing an unmetneed for such therapies. As an example, over-expression ofP-glycoprotein in the BBB of drug-resistant epilepsy patients has beenobserved and about one-third of all epilepsy patients exhibitdrug-resistance.

In a cancer related example, a receptor tyrosine kinase inhibitor calledimatinib has been shown in cell culture studies to be very effective ininhibiting the growth of glioma cells, but the very poor penetration ofthe BBB by imatinib results in very limited efficacy in patients. Theseresearchers showed that the poor brain penetration of imatinib was dueto P-glycoprotein. It should be noted that gliomas make up about 30% ofall brain tumors and nearly four fifths of all malignant brain tumors.

Effective inhibition of P-glycoprotein in the context of thepharmacological penetration of the blood brain barrier by therapeuticsthat are excluded from the brain by the P-glycoprotein component of theBBB would increase the efficacy of these agents. Co-administration ofsuch a P-glycoprotein inhibitor with the normally brain-excluded drugwould allow these drugs to enter the brain.

The methods of the present invention provide for the identification oftargeted inhibition of P-glycoprotein that interrupt or alter either ATPbinding or ATP hydrolysis at the nucleotide binding sites of thetransporter. ATP, adenosine triphosphate, is a compound used in allliving cells. The compound is able to store large amounts of energy andwill release this energy upon hydrolysis of its terminal phosphategroup(s) in order to perform work. P-glycoprotein has two such ATPbinding sites that supply the energy required to perform the work oftransporting a drug transport substrate from one side of the membrane tothe other.

The methods of the present invention allow us to identify potentialinhibitors that only bind to the nucleotide binding domains ofP-glycoprotein and not to the drug binding sites on the protein. Thiseliminates the overarching problem seen with inhibitors that bind to thedrug binding domain. The inhibitor molecules identified with thesemethods only minimally bind to the drug binding and drug transportdomains of P-glycoprotein, and are therefore, only minimally transportedby P-glycoprotein. This means that once the inhibitor molecules areinside the cell, they are not significantly removed from the cell byP-glycoprotein, while at the same time also blocking thechemotherapeutic drug to be exported from the cells resulting ineffective therapy.

Molecular models of human P-glycoprotein and targeted in silicoscreening for inhibitors. Wise described the methodology and results ofmodeling the structural changes in human P-glycoprotein as thetransporter transitions from one conformational state to another duringa catalytic cycle. The computational analyses of structures that thetransporter protein adopts during a drug transport cycle allowed theanalysis of the interactions of millions of drug like molecules with theprotein in distinct catalytically relevant conformations. Such extensivescreens for specific interactions of small molecule compounds withdifferent structural intermediates of P-glycoprotein have not beenpreviously reported.

A subset of the database of commercially available compounds whichincludes molecules with drug-like characteristics was obtained from theZINC website. These compounds were used in in silico docking studiesaimed at identifying drug-like compounds that would interact stronglywith the nucleotide binding domain structures of various humanP-glycoprotein structural conformations. Docking studies were performedwith the Autodock 4.2 program using the high performance computationalfacilities of the Center for Scientific Computing at Southern MethodistUniversity.

To date about 16 million protein-small molecule drug interactions at thenucleotide binding domains of human P-glycoprotein were analyzed.Compounds that showed strong binding interactions to the P-glycoproteinnucleotide binding domain structures were further analyzed for possibleinteractions at the drug binding domains of P-glycoprotein again usingthe Autodock 4.2 programs. Ultimately, the identification of compoundsthat interact strongly at nucleotide binding domains but weakly at thedrug binding domains of P-glycoprotein were sought. This approachidentifies molecules that inhibit the catalytic transport mechanism ofP-glycoprotein by disrupting ATP binding and or hydrolysis but thatwould not be effectively transported out of the cell by virtue of thelack of predicted interactions at the drug-toxin pumping structures ofP-glycoprotein (the drug binding domains).

Several hundred molecules from the ZINC drug-like molecule databasesubset were found that satisfied these hypothetical requirements forP-glycoprotein inhibitors with low probabilities of being transportsubstrates. Of these initial molecules, 35 molecules were purchased fromcommercial sources and used in in vitro biochemical assays to determinethe effectiveness of inhibition of catalysis by P-glycoprotein, and wereused in cell-based assays to estimate toxicity of the compounds on humancells in culture. In addition, assays of sensitization andre-sensitization of human cancer cell lines that either express normallylow amounts of P-glycoprotein or that over-express P-glycoprotein wereperformed. These latter assays actually test the compounds for theirability to overcome multidrug resistances by inhibition of the intrinsicP-glycoprotein transporter in these cancer cells.

The efficacy of molecules identified using in silico methods to functionas potential inhibitors of P-glycoprotein was initially tested withregard to their capability to inhibit ATP hydrolysis by purifiedP-glycoprotein. For this purpose, a well-established activity assay forATP hydrolyzing enzymes that uses the oxidation of NADH to NAD⁺ throughcoupling the reactions of pyruvate kinase and lactate dehydrogenase tothe hydrolysis of ATP was adapted to medium throughput conditions on 96well plates. The decrease of NADH absorption at 340 nm light wasobserved in a BioTek Eon plate reader. In this set-up, the effects ofpotential small molecule inhibitors on the ATP hydrolysis activity ofP-glycoprotein were tested in duplicate or triplicate and were repeatedat least two times using different enzyme preparations.

The P-glycoprotein used in these assays was a close relative of thehuman P-glycoprotein that is found in mice. A mutant version of themouse P-glycoprotein where all intrinsic cysteine amino acids had beenreplaced by alanine residues was recombinantly expressed in the yeastPichia pastoris and used for these initial tests. Purification of theprotein was achieved with small modifications resulting in highlyenriched P-glycoprotein in micelles containing dodecyl maltoside andlysophosphatidyl choline as detergents. Prior to testing theATP-hydrolysis activity, P-glycoprotein was incubated with phosphatidylcholine for stabilization of the enzyme during the assay.

ATP hydrolysis by P-glycoprotein is stimulated by transport substrateslike the calcium channel blocker verapamil which was initially indicatedas an inhibitor of P-glycoprotein. Verapamil inhibits pumping ofchemotherapeutics from cancer cells by competing for the same bindingsites as the chemotherapeutics and therefore is a transport substrateitself. Typically, 150 μM verapamil was added to the assay mixturecontaining purified P-glycoprotein in 96 well plates. Differentconcentrations of the to-be-tested small molecules were added andpre-incubated at 37° C. The ATP hydrolysis reaction was then started byadding equal volume of double concentrated ATPase cocktail. The finalATP concentration in the reaction was 2 mM. The reaction was allowed toproceed for 20 minutes, the rate of ATP hydrolysis was calculated usingthe molar extinction coefficient of NADH, and the one-to-onerelationship of ATP hydrolyzed and NADH oxidized.

We have identified a group of compounds from in silico studies that werepredicted to bind with high affinity at the nucleotide binding domainsbut only weakly at the drug binding domains of P-glycoprotein.Thirty-five molecules were selected as possible inhibitors of the powergenerating functions of P-glycoprotein and were then biochemicallyassayed for inhibition of ATP hydrolysis activity catalyzed byP-glycoprotein. Compounds were initially screened with 25 microMputative inhibitor as described above. Four of the 35 compounds wereobserved to inhibit verapamil-stimulated ATPase activities byP-glycoprotein. These compounds did not significantly stimulate basalATP hydrolysis rates of P-glycoprotein, indicating that they did notinteract with P-glycoprotein drug binding domains (data not shown).

Compounds “SMU-19”, “SMU-29”, “SMU-34” and “SMU-45”, that wereidentified from the in silico screening methods that targetP-glycoprotein nucleotide binding domains and exclude compounds thatinteract strongly with the drug binding structures as described above,were found to significantly inhibit the verapamil-stimulated ATPhydrolysis catalyzed by P-glycoprotein.

One embodiment of the present invention includes a P-glycoproteininhibitory or binding composition having the structure:

The composition is also denoted as Compound SMU-19: methyl4-[bis(2-hydroxy-4-oxochromen-3-yl)methyl]benzoate (ZINC 09973259, CID4694077). However, the composition may include other substitutions ofthe core composition:

where R1, R2, R3, R4, R4′, R5, R5′, R6, R6′, R7, R7′, R8, and R8′ mayindependently be alkyl, alkenyl, alkoxy, carboxy, carboxyl,alkylcarbonyl, alkylcarboxyl, alkanoyloxy, alkoxycarbonyl, or hydroxyl.

Another example of the P-glycoprotein inhibitor composition of theinstant invention

where R1 may be an alkyl (e.g., methyl, ethyl, etc.), R5, and R5′ mayindependently be an alkyl (e.g., methyl, ethyl, etc.), substituted alkyl(e.g., CF₃, CH₂F, etc.), carboxyl, or hydroxyl.

One embodiment of the present invention includes a P-glycoproteininhibitory or binding composition having the structure:

The composition is also denoted as Compound SMU-29:2-[(5-cyclopropyl-1H-1,2,4-triazol-3-yl)sulfanyl]-N-[2-phenyl-5-(2,4,5-trimethylphenyl)pyrazol-3-yl]acetamide(ZINC 08767731, CID 17555821); however, the composition may includeother substitutions of the core composition:

R may be a substituted or unsubstituted C3-C8 monocyclic cycloalkyl, aC3-C8 monocyclic cycloalkenyl, or a 3- to 7-membered monocyclicheterocycle; R1, R1′, R2, R2′, R3, R3′, R4, R4′, R5, and R5′ mayindependently be alkyl, alkenyl, alkoxy, carboxy, carboxyl,alkylcarbonyl, alkylcarboxyl, alkanoyloxy, alkoxycarbonyl, or hydroxyl;R6 may be a C1-C10 alkyl.

One embodiment of the present invention includes a P-glycoproteininhibitory or binding composition having the structure:

The composition is also denoted as Compound SMU-34:2-[1-[4-(4-methoxyphenyl)piperazin-1-yl]-1-oxobutan-2-yl]-4-methyl-[1]benzothiolo[2,3-d]pyridazin-1-one(ZINC 09252021, CID 22514118); however, the composition may includeother substitutions of the core composition:

where R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 may independentlybe alkyl, alkenyl, alkoxy, carboxy, carboxyl, alkylcarbonyl,alkylcarboxyl, alkanoyloxy, alkoxycarbonyl, or hydroxyl.

One embodiment of the present invention includes a P-glycoproteininhibitory or binding composition having the structure:

The composition is also denoted as Compound SMU-45: ethyl1-(1,3-benzodioxole-5-carbonyl)-3-(3-phenylpropyl)piperidine-3-carboxylate(ZINC 15078148, ZINC 15078146, CID 26410703, CID 45252040); however, thecomposition may include other substitutions of the core composition:

where n maybe from 0-10 and R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10may independently be alkyl, alkenyl, alkoxy, carboxy, carboxyl,alkylcarbonyl, alkylcarboxyl, alkanoyloxy, alkoxycarbonyl, or hydroxyl.For example, another embodiment may include the structural formula:

where R6, R7, R8, R9, and R10 may independently be alkyl, alkenyl,alkoxy, carboxy, carboxyl, alkylcarbonyl, alkylcarboxyl, alkanoyloxy,alkoxycarbonyl, or hydroxyl. R may be a substituted or unsubstitutedC3-C8 monocyclic cycloalkyl, a C3-C8 monocyclic cycloalkenyl, or a 3- to7-membered monocyclic heterocycle; and optionally connected by an alkyl,alkenyl, alkoxy, carboxy, carboxyl, alkylcarbonyl, alkylcarboxyl,alkanoyloxy, alkoxycarbonyl, or hydroxyl; R6 may be a C1-C10 alkyl.

These P-glycoprotein inhibitors were investigated further. FIGS. 1A-1Dshow the ATP hydrolysis by P-glycoprotein in the presence of varyingconcentrations of inhibitor compounds normalized to verapamil-stimulatedP-glycoprotein activity. IC50 values for the inhibitors in these assayswere observed to be about 20 microM.

FIGS. 1A-1D are plots showing the inhibition of ATP hydrolysis by insilico identified compounds. The FIG. shows the concentration dependenceof inhibition of the four identified inhibitor compounds (abbreviated19, 29, 34 and 45). Each plot represents the composite results of 3 to 5individual studies performed on three different P-glycoproteinpreparations.

Determination of the possible mode of ATP hydrolysis inhibition by theidentified P-glycoprotein inhibitors. The mode of hydrolysis of ATP byP-glycoprotein was investigated by measuring the maximal bindingstoichiometry and binding affinity of an analog of ATP, SL-ATP, and byElectron Spin Resonance (ESR) spectroscopy, similar to in both thepresence and absence of the identified inhibitors.

ESR spectra are acquired for known concentrations of SL-ATP and comparedto spectra acquired in the presence of known concentrations ofP-glycoprotein, verapamil and inhibitor molecule. The high-field signalof the freely mobile, unbound SL-ATP is compared and is used tocalculate the amount of ATP-analog bound to P-glycoprotein. The resultsof these studies are presented in FIG. 2. Binding of SL-ATP in theabsence of any P-glycoprotein inhibitor saturated at about 2 mol ofSL-nucleotide bounds per mol of P-glycoprotein.

FIGS. 2A-2D are plots showing the effects of P-glycoprotein ATPhydrolysis inhibitors on nucleotide binding. Between 30 and 50 microMPgp were incubated with 50 microM inhibitor compound. Increasing amountsof SL-ATP were added. The ESR signal of the free SL-ATP in the presenceof P-glycoprotein and inhibitor was compared to that in the absence ofP-glycoprotein. The graphs represent at least 3 individual studies eachusing 3 to 5 different P-glycoprotein preparations.

Compounds SMU-19 and SMU-45 reduced the stoichiometry of binding ofSL-nucleotides to P-glycoprotein. Compounds SMU-29 and SMU-34 decreasedthe SL-ATP binding affinities for P-glycoprotein. All four of theinhibitor compounds effected SL-ATP binding, which provides physicalevidence that the in silico selection for binding of inhibitor at thenucleotide binding domains of P-glycoprotein worked as hypothesized forthese compounds.

FIGS. 3A-3B are plots that show an example of the re-sensitization of amultidrug resistant prostate cancer cell line to two differentchemotherapeutics using the prostate cancer derived cell line, DU145 andthe multidrug-resistant variant, DU145-TxR. Addition of 25 μM compound29, 34 or 45 restores chemotherapeutic sensitivity of MDR prostatecancer cell line DU145-TxR to that of nonresistant parental DU145. Cellviability was measured by the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assayconducted as described previously. After 48 hours of treatment withPaclitaxel or Doxorubicin alone or in the presence of 25 μM compound 29,34, or 45 media was removed and cells were washed twice with phosphatebuffered saline (PBS) and MTT assay was conducted 24 hours later. Dataexpressed as mean±SEM.

Compound SMU-19 was found to not affect multidrug resistance in thesecell culture studies and was not included in the FIG. 2A. Cell viabilitywas measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay conducted as described previously (36).

FIGS. 3A-3B clearly demonstrate that compounds SMU-29, SMU-34 and SMU-45reverse the Paclitaxel resistance phenotype of the DU145TxR to drugsensitivity levels that are equivalent to the non-drug resistant DU145parent cell lines (left panels). Studies testing the re-sensitization ofthe drug resistant cells to a different chemotherapeutic, doxorubicin(right panels), suggest that the reversal of Paclitaxel resistancereflects a general reversal of multidrug resistance, since doxorubicinresistance by DU145TxR is also reversed to levels equivalent to thenon-resistant DU145 parental strain.

Hyper-sensitization of cancers to chemotherapeutics. Other studies (notshown) using the ovarian cancer cell lines A2780 indicated that thepresence of compounds SMU-29, SMU-34 or SMU-45 hyper-sensitized thecells to doxorubicin. The sensitivity of normal, not chemotherapyresistant A2780 ovarian cancer cell lines to doxorubicin was increasedby several orders of magnitude in these assays.

Toxicity analysis. Toxicity studies using non-cancerous cells suggestedlow overall toxicity of the compounds by themselves at the sameconcentrations of compounds where reversal of chemotherapy resistance orhyper-sensitization of ovarian cancer cell lines was observed (notshown).

Cell Culture Assays.

FIGS. 4A and 4B show the in silico identified P-gp inhibitors potentiatethe cytotoxic effects of paclitaxel in the MDR human prostate cancercell line DU145TxR. Cells were incubated with 50 nM-25 μM inhibitor 19(diamonds), 29 (squares), 34 (triangles), 45 (inverted-triangles), orverapamil (octagons) with 500 nM paclitaxel (top) or without paclitaxel(bottom) for 48 hours and survival was determined by MTT assay aspreviously described. Values represent the mean±SEM of at least twoseparate experiments performed in triplicate.

A range of experimental inhibitor concentrations was added in thepresence of 500 nM paclitaxel and the cell viability was assessed usingthe MTT assay. The results shown in FIG. 4A indicate that three of thefour compounds, compounds 29 (squares), 34 (triangles) and 45 (invertedtriangles) strongly affected the sensitivity of the resistant DU145TxRcells to paclitaxel at that concentration, similar to the effects ofverapamil (octagons), a known resistance-modifying agent and competitiveinhibitor of P-gp drug transport (Yusa and Tsuruo, 1989). Compound 19(diamonds) had no effect on paclitaxel sensitivity of DU145TxR. To testwhether the loss in cell viability may be caused by the compoundsthemselves, similar studies as above were performed in the absence ofpaclitaxel, FIG. 4B. The results clearly showed that there-sensitization effect by compounds 29, 34 and 45 was likely not aresult of intrinsic toxicity of P-gp inhibitors at testedconcentrations, since 1 μM of inhibitors reduced survival by less than20% in the absence of paclitaxel.

In order to compare the efficacy of inhibitors, the potentiationconcentration of inhibitor resulting in 50% reduction in cell viability,PC₅₀, in the presence of 500 nM paclitaxel was calculated from the datain FIG. 4A. The PC₅₀ of compounds 29, 34, 45 were calculated to be2.33±0.01 μM, 0.57±0.003 μM and 2.0±0.01 μM respectively, very similarto the known inhibitor, verapamil, with a PC₅₀ of 0.86±0.01 μM. The PC₅₀for compound 19 was not determined, as it seemed to have no effect onthe toxicity of paclitaxel in these experiments.

FIGS. 5A to 5C show the intrinsic toxicities of experimental compounds:The effects of compounds 19 (diamonds), 29 (squares), 34 (triangles), 45(inverted triangles) and verapamil (octagons) on the survival of (5A)noncancerous HFL1, (5B) prostate cancer cell line, DU145, (5C) MDRprostate cancer cell line, DU145TxR. Percent survival determined by MTTassay calculated relative to cells treated with equal volume DMSOvehicle and represent the mean±SEM from 2-4 separate studies, each studyperformed in triplicate wells.

The intrinsic in vitro toxicities of identified compounds were evaluatedto determine their potential therapeutic window. This was assessed in anoncancerous cell line (HFL1) and the human prostate cancer cell linesDU145 and the MDR sub-line DU145TxR. The potential toxicity of thecompounds were assessed at concentrations that centered around 25 μM. Atthis concentration the compounds were shown to significantly inhibit ATPhydrolysis in biochemical assays with purified P-gp (Brewer et al.,2014). The presence of 25 μM of compounds 29, 34 and 45 also causedclose to full re-sensitization of the multidrug resistant DU145TxR cellsto paclitaxel as shown in FIG. 5C. In vitro cytotoxic concentrationsdetermined using the HFL1 cell line have been shown to be comparable towhole animal toxicity testing as predictors of human toxicity (Barileand Cardona, 1998; Yang et al., 2002). The three cell lines, HFL1, DU145and DU145TxR were exposed to verapamil (octagons), compound 19(diamonds), 29 (squares), 34 (triangles) and 45 (inverted triangles) for48 hours and survival was determined using the MTT assay. The IC₅₀values are summarized in Table 1 and were calculated from graphs shownin FIG. 2A-C.

TABLE 1 IC₅₀ (μM) concentration for P-gp inhibitors ± standard error.Values were calculated from the mean of 2-4 separate experimentsperformed in triplicate wells. IC₅₀ (μM) concentration for P-gpinhibitors ± standard error. Values were calculated from the mean of 2-4separate experiments performed in triplicate wells. ND, not determinedND, not determined. IC₅₀ (μM) SMU-19 SMU-29 SMU-34 SMU-45 Verapamil HFL1780.3 ± 0.37 48.2 ± 0.01 85.2 ± 0.04 60.3 ± 0.02 ND DU145 106.3 ± 0.1033.4 ± 0.01 47.2 ± 0.02 41.2 ± 0.03 74.5 ± 0.04 DU145TxR ND 37.9 ± 0.02200.3 ± 0.17  99.3 ± 0.08 147.5 ± 0.10 

FIGS. 6A to 6C show the dose dependent sensitization of MDR prostatecancer cell line DU145TxR. DU145TxR cells (closed symbols) wereincubated with various concentrations of paclitaxel and 5 μM (FIG. 6A),10 μM (FIG. 6B), or 25 μM (FIG. 6C) in silico identified P-gp inhibitors19 (diamonds), 29 (squares), 34 (triangles), 45 (inverted triangles) andverapamil (octagons). For reference, the DU145TxR (closed circle) andDU145 (open circle) incubated with paclitaxel only are also included.Values are mean±SEM from at least 2 separate experiments performed intriplicate wells. The data were normalized to the intrinsic toxicitiesas determined in studies from FIGS. 5A to 5C of each of the compoundsfor each of the corresponding concentrations.

Dose Dependent Sensitization of MDR Prostate Cancer Cell Line: Todetermine the degree to which the MDR prostate cancer cell line DU145TxRcould be sensitized to paclitaxel the inventors determined cell survivalat increasing concentrations of paclitaxel in the presence of threeconcentrations of P-gp inhibitors. FIGS. 6A, 6B and 6C show the survivalof DU145TxR at increasing concentration of paclitaxel in the presence of5 μM, 10 μM and 25 μM of the experimental compounds, respectively. Theopen circles represent the parental DU145 cell line in the presence ofpaclitaxel without addition of any inhibitor compound. The closedcircles represent the survival of the multidrug resistant DU145TxR inthe presence of paclitaxel alone. Diamonds represent the presence ofcompound 19, squares represent the presence of compound 29, trianglesrepresent compound 34, inverted triangles compound 45 and octagonsrepresent the presence of verapamil. The data indicate that increasedsensitization of DU145TxR is already observed in the presence of 5 μM ofthe experimental compounds, 29, 34 and 45. At that concentration over10-fold increase in paclitaxel sensitivity in DU145TxR is observedcompared to DU145TxR without P-gp inhibitors. Doubling the concentrationof the compounds to 10 μM approximately doubled the DU145TxR sensitivityto paclitaxel. At both concentrations (5 μM and 10 μM) the effects ofthe compounds were comparable to those of the known MDR modulator,verapamil, while compound 19 showed no effect on cell viability. At 25μM concentration, compounds 29 and 34 sensitized the multidrug resistantDU145TxR to IC₅₀ values that were close to those observed for theparental, sensitive DU145 cell line (open circles), an 800- or 1200-foldsensitization, respectively. Sensitization of DU145TxR to paclitaxel bythe compounds 29 (squares), 34 (triangles) and 45 (inverted triangles)exceeded sensitization by verapamil (octagons). Compound 19 did not showany effect on cell viability in any of the studies at any concentration(diamonds).

Extended biophysical evaluations by the present inventors are providedin their article “In Silico Screening for Inhibitors of P-GlycoproteinThat Target the Nucleotide Binding Domains” (Brewer et al., 2014), MolPharmacol 86:716-726, December 2014, the entirety of which isincorporated herein by reference. Brewer et al., in FIGS. 4 and 5,provide additional information about the mechanism of inhibitor actionthat was predicted computationally was confirmed using electron spinresonance spectroscopy (ESR) and titration of accessible nucleotidebinding sites. Inhibitors 19, 34 and 45 were predicted to interactdirectly with the nucleotide binding sites (FIG. 5 of Brewer). The ESRstudies of Brewer et al., show that these three compounds interferedirectly with the nucleotide binding sites in as they reduce the numberof binding sites that are able to bind an ATP analog, see FIGS. 4C, 4Eand 4F of Brewer (incorporated herein by reference). Compound 29 waspredicted computationally to bind outside of the nucleotide bindingssites (FIG. 5 of Brewer) and was shown to not interfere with ATP bindingin ESR experiments, see FIG. 4D of Brewer.

The present inventors have successfully identified a number of leadcompounds that inhibit the power-stroke of drug expulsion catalyzed bythe multidrug resistance P-glycoprotein. These results show thateffective inhibition of P-glycoprotein can occur by targeting thenucleotide binding domains of the protein that can causere-sensitization of multidrug resistant cancer cell lines. Throughbiophysical assays the inventors determined additional non-limitinginformation about the mechanism of P-glycoprotein inhibition by theidentified compounds. The identified P-glycoprotein inhibitor leadcompounds are specific inhibitors that can be co-administered withtraditional chemotherapeutics to treat chemotherapy resistant cancers ormultidrug resistant infections. The identified P-glycoprotein inhibitorcompounds sensitize cancer stem cells to chemotherapeutic treatment,removing these cells as a source of recurrent cancer. The identifiedP-glycoprotein inhibitor lead compounds are specific inhibitors that canbe co-administered with therapeutics that do not normally penetrate theblood brain barrier. This increases the efficacy of drugs when access tothe central nervous system by the drug is prohibited by P-glycoprotein.

Co-administration with chemotherapeutic and/or anti-cancer agents. Inaccordance with the methods of the invention, the compositions of thepresent invention can be co-administered in combination with anti-canceragents (“anticancer agent” or “chemotherapeutic agent”). Withoutintending to be bound by any particular mechanism or effect, suchco-administration can in some cases provide one or more of severalunexpected benefits including: (i) co-administration of the compositionsof the present invention and the chemotherapeutic agent has asynergistic effect on induction of cancer cell death; (ii)co-administration provides a better therapeutic result thanadministration of the chemotherapeutic agent alone, e.g., greateralleviation or amelioration of one or more symptoms of the cancer,diminishment of extent of disease, delay or slowing of diseaseprogression, amelioration, palliation or stabilization of the diseasestate, partial or complete remission, prolonged survival or otherbeneficial therapeutic results; (iii) co-administration of thecompositions of the present invention increases the sensitivity ofcancer cells to the anticancer agent, allowing lower doses of the agentto be administered to the patient or allowing an agent to be used fortreatment of cells otherwise resistant to the agent or otherwiserefractory to treatment; and (iv) co-administration of the compositionsof the present invention and the chemotherapeutic agent increaseskilling of cells in hypoxic regions of tumors that are not efficientlykilled by the agent alone.

Examples of chemotherapeutic and/or anti-cancer agents include agentssuch as paclitaxel, doxorubicin, vincristine, vinblastine, vindesine,vinorelbin, taxotere (DOCETAXEL), topotecan, camptothecin, irinotecanhydrochloride (CAMPTOSAR), etoposide, mitoxantrone, daunorubicin,idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantronehydrochloride, 5-fluorouracil, methotrexate, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, cytarabine (ARA-C), trimetrexate,gemcitabine, acivicin, alanosine, pyrazofurin,N-Phosphoracetyl-L-Asparate (PALA), pentostatin, 5-azacitidine,5-Aza-2′-deoxycytidine, adenosine arabinoside (ARA-A), cladribine,ftorafur, UFT (combination of uracil and ftorafur),5-fluoro-2′-deoxyuridine, 5-fluorouridine, 5′-deoxy-5-fluorouridine,hydroxyurea, dihydrolenchlorambucil, tiazofurin, cisplatin, carboplatin,oxaliplatin, mitomycin C, BCNU (Carmustine), melphalan, thiotepa,busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate,cyclophosphamide, nitrogen, mustard, uracil mustard, pipobroman,4-ipomeanol, dihydrolenperone, spiromustine, geldanamycin,cytochalasins, depsipeptide, Lupron, ketoconazole, tamoxifen, goserelin(Zoledax), flutamide,4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluorometh-yl)propionanilide, Herceptin, anti-CD20 (Rituxan), interferon, alpha,interferon beta, interferon gamma, interleukin 2, interleukin 4,interleukin 12, tumor necrosis factors, and radiation.

The disclosed method is particularly effective at treating subjectswhose cancer has become “multi-drug resistant”. A cancer which initiallyresponded to an anti-cancer drug becomes resistant to the anti-cancerdrug when the anti-cancer drug is no longer effective in treating thesubject with the cancer. For example, many tumors will initially respondto treatment with an anti-cancer drug by decreasing in size or evengoing into remission, only to develop resistance to the drug. Drugresistant tumors are characterized by a resumption of their growthand/or reappearance after having seemingly gone into remission, despitethe administration of increased dosages of the anti-cancer drug. Cancersthat have developed resistance to two or more anti-cancer drugs are saidto be “multi-drug resistant”. For example, it is common for cancers tobecome resistant to three or more anti-cancer agents, often five or moreanti-cancer agents and at times ten or more anti-cancer agents.

Another embodiment of the present invention is a method of treating asubject with a cancer that has become drug resistant. Optionally, themethod of the invention can be used for a multi-drug resistant cancer.The method comprises the step of administering an effective amount of acompound of the present invention, pharmaceutically acceptable salt,solvate, clathrate, or a prodrug thereof. Preferably, one or moreadditional anti-cancer drugs are co-administered with a compound of theinvention. Examples of anti-cancer drugs are described below. Forexample, the co-administered anti-cancer drug is an agent thatstabilizes mictotubules, such as TAXOL or a taxanes derivative.

In another embodiment, a compound of the invention can be administeredas adjuvant therapy to prevent the reoccurrence of cancer. For example,stage II and stage III melanoma are typically treated with surgery toremove the melanoma followed by chemotherapeutic treatment to preventthe reoccurrence of cancer. In one embodiment, one or more additionalanti-cancer drugs are co-administered with a compound of the inventionas adjuvant therapy. Examples of anti-cancer drugs are described herein.In one embodiment, the co-administered anti-cancer drug is an agent thatstabilizes microtubules, such as TAXOL or a taxanes derivative. Inanother embodiment, the co-administered anti-cancer drug is animmunotherapeutic anticancer agent.

Drug resistant cancers that can be treated or prevented by the methodsof the present invention include, but are not limited to human sarcomasand carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, anal carcinoma,esophageal cancer, gastric cancer, hepatocellular cancer, bladdercancer, endometrial Cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, stomach cancer, atrial myxomas, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, thyroid and parathyroid neoplasms, papillarycarcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'tumor, cervical cancer, testicular tumor, lung carcinoma, small celllung carcinoma, non-small-cell lung cancer, bladder carcinoma,epithelial carcinoma, glioma, pituitary neoplasms, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, schwannomas, oligodendroglioma,meningioma, spinal cord tumors, melanoma, neuroblastoma,pheochromocytoma, Types 1-3 endocrine neoplasia, retinoblastoma;leukemias, e.g., acute lymphocytic leukemia and acute myelocyticleukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia); chronic leukemia (chronic myelocytic (granulocytic)leukemia and chronic lymphocytic leukemia); and polycythemia vera,lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiplemyeloma, Waldenstrobm's macroglobulinemia, and heavy chain disease.Other examples of leukemias include acute and/or chronic leukemias,e.g., lymphocytic leukemia (e.g., as exemplified by the p388 (murine)cell line), large granular lymphocytic leukemia, and lymphoblasticleukemia; T-cell leukemias, e.g., T-cell leukemia (e.g., as exemplifiedby the CEM, Jurkat, and HSB-2 (acute), YAC-1 (murine) cell lines),T-lymphocytic leukemia, and T-lymphoblastic leukemia; B cell leukemia(e.g., as exemplified by the SB (acute) cell line), and B-lymphocyticleukemia; mixed cell leukemias, e.g., B and T cell leukemia and B and Tlymphocytic leukemia; myeloid leukemias, e.g., granulocytic leukemia,myelocytic leukemia (e.g., as exemplified by the HL-60 (promyelocyte)cell line), and myelogenous leukemia (e.g., as exemplified by theK562(chronic) cell line); neutrophilic leukemia; eosinophilic leukemia;monocytic leukemia (e.g., as exemplified by the THP-1(acute) cell line);myelomonocytic leukemia; Naegeli-type myeloid leukemia; andnonlymphocytic leukemia. Other examples of leukemias are described inChapter 60 of The Chemotherapy Sourcebook, Michael C. Perry Ed.,Williams & Williams (1992) and Section 36 of Holland Frie CancerMedicine 5th Ed., Bast et al. Eds., B. C. Decker Inc. (2000). The entireteachings of the preceding references are incorporated herein byreference.

Additional drug resistant cancers that can be treated or prevented bythe methods of the present invention include, but are not limited tooral cavity and pharynx cancers, including tongue, mouth, pharynx, andother oral cavity cancers; digestive system cancers, includingesophagus, small intestine, rectum, anus, anal canal, anorectum, liverand intrahepatic bile duct, gallbladder and other biliary, pancreas andother digestive organs; respiratory system cancers, including larynx andbronchus; bone and joint cancers; soft tissue (including heart) cancers;genital system cancers, including uterine cervix, uterine corpus, ovary,vulva, vagina and other genital, female, testis, penis and othergenital, male; urinary system cancers, including kidney and renalpelvis, and ureter and other urinary organs; eye and orbit cancers;leukemia, including acute myeloid leukemia and chronic myeloid leukemia.

Numerous non-cancer diseases that involve excessive orhyperproliferative cell growth, termed hyperplasia that have become drugresistant can be treated with the instant compositions. Non-cancerousproliferative disorders include smooth muscle cell proliferation,systemic sclerosis, cirrhosis of the liver, adult respiratory distresssyndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy,e.g., diabetic retinopathy or other retinopathies, cardiac hyperplasia,reproductive system associated disorders such as benign prostatichyperplasia and ovarian cysts, pulmonary fibrosis, endometriosis,fibromatosis, harmatomas, lymphangiomatosis, sarcoidosis, desmoid tumorsand the like.

The present disclosure provides a method of treating a subject havingcells that are resistant to one or more drugs by identifying a subjecthaving one or more drug resistant cells; administering to the subject apharmaceutically effective amount of an inhibitor compound having one ofthe following structural formulas:

andcontacting one or more drug resistant cells with the inhibitor compoundto reduce the export of the inhibitor compound from the one or more drugresistant cells and to block the transport of chemotherapeutic drug(s)from the one or more drug resistant cancer cells.

The inhibitor compound interacts with an exporter protein. In oneembodiment, the inhibitor compound interacts with drug-toxin pumpingstructures of a P-glycoprotein. In one embodiment, the inhibitorcompound interacts with ATP binding domain(s) of a P-glycoprotein andthe inhibitor compound does not bind to drug binding site(s) on theP-glycoprotein. In one embodiment, the inhibitor compound is aP-glycoprotein inhibitor. The inhibitor compound is minimallytransported by a P-glycoprotein.

The one or more drug resistant cancer cells may be one or more multidrugresistant tumor cells, cancer cells, cancer stem cells, bacterial cells,virus infected cells and the like.

The method may further include the step of administering one or morechemotherapeutic agents to the subject before further treatment, duringtreatment or after treatment with the inhibitor compound.

The inhibitor compound is effective for increasing the effectiveness ofthe chemotherapeutic drug or antibiotic agents to inhibit proliferation,inducing cell death, or indirectly inhibiting development of a tumor bysuppressing tumor angiogenesis.

The inhibitor compound also is effective for increasing an efficacy ofone or more chemotherapeutics or antibiotic agents and/or decreasingtoxicity of the chemotherapeutic treatment(s) or antibiotic treatments.

The present invention encompasses all compounds having identicalmolecular formula but differing in the nature or sequence of bonding oftheir atoms or in the arrangement of their atoms in space. A carbon atombonded to four nonidentical substituents is termed a “chiral center” anda compound with one chiral center has two enantiomeric forms of oppositechirality. It is contemplated that any embodiment discussed in thisspecification includes compositions having one or more centers ofchirality and exist as stereochemically isomeric forms. The presentinvention encompasses all isomers of the compositions disclosed herein.As used herein “isomers” denote differ arrangements of atoms in spaceand include “stereoisomers”. Stereoisomers that are not mirror images ofone another are termed “diastereomers” and stereoisomers that arenonsuperimposable mirror images are termed “enantiomers” or sometimes“optical isomers.” The present invention encompasses all variations ateach of these chiral centers to include numerous composition having thesame formula but different arrangement of their atoms in space.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), propertie(s), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skill in the art recognize themodified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

-   Bartle, F. A., and Cardona, M. (1998). Acute cytotoxicity testing    with cultured human lung and dermal cells. In vitro cellular &    developmental biology Animal 34, 631-635.-   Brewer, F. K., Follit, C. A., Vogel, P. D., and Wise, J. G. (2014).    In silico Screening for Inhibitors of P-Glycoprotein that Target the    Nucleotide Binding Domains. Molecular pharmacology 86, 716-726.-   Yang, A., Cardona, D. L., and Bartle, F. A. (2002). Subacute    cytotoxicity testing with cultured human lung cells. Toxicology in    vitro: an international journal published in association with BIBRA    16, 33-39.-   Yusa, K., and Tsuruo, T. (1989). Reversal mechanism of multidrug    resistance by verapamil: direct binding of verapamil to    P-glycoprotein on specific sites and transport of verapamil outward    across the plasma membrane of K562/ADM cells. Cancer Res 49,    5002-5006.

What is claimed is:
 1. A method of treating a subject having cancer thatis resistant to one or more chemotherapeutic drugs comprising the stepsof: identifying a subject having one or more drug resistant cancer cellsthat express a P-glycoprotein; administering to the subject apharmaceutically effective amount of an inhibitor compound having one ofthe following structural formulas:

and contacting one or more drug resistant cancer cells with theinhibitor compound to reduce the drug resistance of the cancer cells. 2.The method of claim 1, wherein the inhibitor compound interacts with anexporter protein.
 3. The method of claim 1, wherein the inhibitorcompound is a P-glycoprotein inhibitor.
 4. The method of claim 1,wherein the inhibitor compound interacts with drug-toxin pumpingstructures of a P-glycoprotein.
 5. The method of claim 1, wherein theinhibitor compound interacts with ATP binding domain(s) of aP-glycoprotein and the inhibitor compound does not bind to drug bindingsite(s) on the P-glycoprotein.
 6. The method of claim 1, wherein theinhibitor compound is transported by a P-glycoprotein.
 7. The method ofclaim 1, wherein the one or more drug resistant cancer cells are one ormore multidrug resistant tumor cells.
 8. The method of claim 1, whereinthe one or more drug resistant cancer cells are lymphoma, leukemia,cancer stem cells, nonsmall-cell lung cancer, liver cancer, encephaloma,leukocythemia, carcinoma of prostate, intestine cancer, myeloma tumor,lymphoma, breast carcinoma, ovarian cancer, gastric cancer, small celllung cancer, esophageal carcinoma, esophageal carcinoma, and sarcoma. 9.The method of claim 1, further comprising the step of administering oneor more chemotherapeutic agents to the subject.
 10. The method of claim1, wherein the inhibitor compound is effective for at least one of:increasing the effectiveness of the chemotherapeutic drug to inhibitproliferation, inducing cell death, or indirectly inhibiting developmentof a tumor by suppressing tumor angiogenesis, reducing the export of theinhibitor compound from the one or more drug resistant tumor cells, toblock the transport of chemotherapeutic drug(s) from the one or moredrug resistant cancer cells, or increasing an efficacy of one or morechemotherapeutics and/or decreasing toxicity of the chemotherapeutictreatment(s).
 11. A method of sensitization and re-sensitization of acancer cell to a chemotherapeutic comprising the steps of: identifying asubject having one or more cancer cells that express P-glycoprotein inneed of sensitization and re-sensitization to one or morechemotherapeutic; administering to the subject a pharmaceuticallyeffective amount of an inhibitor compound having one of the followingstructural formulas:

and contacting one or more cancer cells with the inhibitor compound toreduce the export of the inhibitor compound from the one or more cancercells and to block the transport of the one or more chemotherapeuticdrug(s) from the one or more cancer cells to sensitization orre-sensitization of the one or more cancer cells to the one or morechemotherapeutic drug(s).
 12. The method of claim 11, wherein theinhibitor compound is at least one of a P-glycoprotein inhibitor, aninhibitor compound interacts with one or more ATP binding domain(s) of aP-glycoprotein, or an inhibitor compound that does not bind to drugbinding sites on the P-glycoprotein.
 13. The method of claim 11, whereinthe one or more drug resistant cancer cells are one or more multidrugresistant tumor cells or cancer stem cells.
 14. The method of claim 11,further comprising the step of administering one or morechemotherapeutic agents to the subject.
 15. A method of increasing anefficacy of one or more chemotherapeutics and/or decreasing toxicity ofone or more chemotherapeutic treatments comprising the steps of:identifying a subject having one or more cancer cells that express aP-glycoprotein; administering to the subject one or morechemotherapeutics; administering to the subject a pharmaceuticallyeffective amount of an inhibitor compound having one of the followingformulas:

and contacting one or more cancer cells with the inhibitor compound toreduce the export of the inhibitor compound from the one or more cancercells and to block the transport of the one or more chemotherapeuticsfrom the one or more cancer cells to increasing the efficacy of the oneor more chemotherapeutics and/or decreasing toxicity of the one or morechemotherapeutic treatments.
 16. The method of claim 15, wherein theinhibitor compound is at least one of: a P-glycoprotein inhibitor, a Pinhibitor compound interacts with one or more ATP binding domains of aP-glycoprotein, a P-glycoprotein inhibitor compound does not bind todrug binding site(s) on the P-glycoprotein, or an inhibitor compoundthat is minimally transported by a P-glycoprotein.
 17. The method ofclaim 15, wherein the one or more drug resistant cancer cells are one ormore multidrug resistant cancer cells.
 18. A method of increasing thepenetration of an agent through the blood brain barrier or the bloodtestis barrier comprising the steps of: identifying a subject in need ofincreasing the penetration of an agent through the blood brain barrieror blood testis barrier; administering to the subject a pharmaceuticallyeffective amount of an inhibitor compound having one of the followingstructural formulas:

and wherein the inhibitor compound reduces the export of the inhibitorcompound from the one or more cells and increases the penetration of theagent through the blood brain barrier or the blood testis barrier. 19.The method of claim 18, wherein the inhibitor compound is at least oneof a P-glycoprotein inhibitor, an inhibitor compound interacts with oneor more ATP binding domains of a P-glycoprotein and the P-glycoproteininhibitor compound does not bind to drug binding site(s) on theP-glycoprotein, or an inhibitor compound that is minimally transportedby a P-glycoprotein.
 20. A method of decreasing the P-glycoproteinactivity of a cancer patient who has built up resistance to atherapeutically active agent used in the treatment of a cancer thatexpresses a P-glycoprotein to reduce the resistance to further treatmentwith the substance comprising the steps of: administering to a cancerpatient before further treatment, during treatment or after treatmentwith the therapeutically active agent, an amount of an inhibitorcompound having one of the following structural formulas:

to reduce the export of the inhibitor compound and to block thetransport of the therapeutically active agent from one or more cancercells of the cancer patient.
 21. A method of reducing the likelihood ofdeveloping a resistance to a therapeutically active agent comprising thesteps of: administering to a patient before further treatment, duringtreatment or after treatment with the therapeutically active agent, anamount of an inhibitor compound having one of the following structuralformulas:

to reduce the export of the inhibitor compound and to block thetransport of the therapeutically active agent from one or more cells ofthe patient.
 22. The method of claim 21, wherein the inhibitor compoundis a P-glycoprotein inhibitor.
 23. The method of claim 21, wherein thetherapeutically active agent is an antibiotic.
 24. A method formodulating the activity of a cell membrane transporter in a biologictissue comprising the steps of: contacting a tissue having a cellmembrane transporter with a pharmaceutically effective amount of ainhibitor compound having one of the following structural formulas:


25. The method of claim 24, wherein the inhibitor compound is aP-glycoprotein inhibitor.
 26. A method for treating a disease orcondition in a mammal or avian resulting from an activity of a cellmembrane transporter comprising the steps of: administering to saidmammal or avian a therapeutically effective amount of a pharmaceuticalcomposition having the structural formula:

or pharmaceutically acceptable salts thereof and a pharmaceuticallyacceptable excipient.